This invention relates to polypeptide hormone analogues that contain a glucose-conformational switch and so exhibit glucose-responsive rates of hormone disassembly or glucose-responsive binding to cognate cellular receptors. Application to insulin is described in relation to the treatment of patients and non-human mammals with Type 1 or Type 2 diabetes mellitus by subcutaneous, intraperitoneal or intravenous injection. The insulin analogues of the present invention may also exhibit other enhanced pharmaceutical properties, such as increased thermodynamic stability, augmented resistance to thermal fibrillation above room temperature, decreased mitogenicity, and/or altered pharmacokinetic and pharmacodynamic properties. More particularly, this invention relates to insulin analogues that confer either rapid action (relative to wild-type insulin in its regular soluble formulation), intermediate action (comparable to NPH insulin formulations known in the art) or protracted action (comparable to basal insulins known in the art as exemplified by insulin detemir and insulin glargine) such that the affinity of the said analogues for the insulin receptor is higher when dissolved in a solution containing glucose at a concentration above the physiological range (>140 mg/dl; hyperglycemia) than when dissolved in a solution containing glucose at a concentration below the physiological range (<80 mg/dl; hypoglycemia).
The engineering of non-standard proteins, including therapeutic agents and vaccines, may have broad medical and societal benefits. Naturally occurring proteins as encoded in the genomes of human beings, other mammals, vertebrate organisms, invertebrate organisms, or eukaryotic cells in general—may have evolved to function optimally within a cellular context but may be suboptimal for therapeutic applications. Analogues of such proteins may exhibit improved biophysical, biochemical, or biological properties. A benefit of protein analogues would be to achieve enhanced activity (such as metabolic regulation of metabolism leading to reduction in blood-glucose concentration under conditions of hyperglycemia) with decreased unfavorable effects (such as induction of hypoglycemia or its exacerbation). An example of a therapeutic protein is provided by insulin. Wild-type human insulin and insulin molecules encoded in the genomes of other mammals bind to insulin receptors is multiple organs and diverse types of cells, irrespective of the receptor isoform generated by alternative modes of RNA splicing or by alternative patterns of post-translational glycosylation. An example of a medical benefit would be the non-standard design of a soluble insulin analogue whose intrinsic affinity for insulin receptors on the surface of target cells, and hence whose biological potency, would depend on the concentration of glucose in the blood stream.
The insulin molecule contains two chains, an A chain, containing 21 residues, and a B chain containing 30 residues. The mature hormone is derived from a longer single-chain precursor, designated proinsulin, as outlined in FIG. 1. Specific residues in the insulin molecule are indicated by the amino-acid type (typically in standard three-letter code; e.g., Lys and Ala indicate Lysine and Alanine) and in superscript the chain (A or B) and position in that chain. For example, Alanine at position 14 of the B chain of human insulin is indicated by AlaB14; and likewise Lysine at position B28 of insulin lispro (the active component of Humalog®; Eli Lilly and Co.) is indicated by LysB28. Although the hormone is stored in the pancreatic β-cell as a Zn2+-stabilized hexamer, it functions as a Zn2+-free monomer in the bloodstream. Administration of insulin has long been established as a treatment for diabetes mellitus. A major goal of conventional insulin replacement therapy in patients with diabetes mellitus is tight control of the blood glucose concentration to prevent its excursion above or below the normal range characteristic of healthy human subjects. Excursions above the normal range are associated with increased long-term risk of microvascular disease, including retinapathy, blindness, and renal failure. Hypoglycemia in patients with diabetes mellitus is a frequent complication of insulin replacement therapy and when severe can lead to significant morbidity (including altered mental status, loss of consciousness, seizures, and death). Indeed, fear of such complications poses a major barrier to efforts by patients (and physicians) to obtain rigorous control of blood glucose concentrations (i.e., excusions within or just above the normal range), and in patients with long-established Type 2 diabetes mellitus such efforts (“tight control”) may lead to increased mortally. In addition to the above consequences of severe hypoglycemia (designated neuroglycopenic effects), mild hypoglycemia may activate counter-regulatory mechanisms, including over-activation of the sympathetic nervous system leading to turn to anxiety and tremulousness (symptoms designated adrenergic). Patients with diabetes mellitus may not exhibit such warning signs, however, a condition known as hypoglycemic unawareness. The absence of symptoms of mild hypoglycemia increases the risk of major hypoglycemia and its associated morbidity and mortality. Multiple and recurrent episodes of hypoglycemia are also associated with chronic cognitive decline, a proposed mechanism underlying the increased prevalence of dementia in patients with long-standing diabetes mellitus. There is therefore an urgent need for new diabetes treatment technologies that would reduce the risk of hypoglycemia while preventing upward excursions in blood-glucose concentration above the normal range.
Diverse technologies have been developed in an effort to mitigate the threat of hypoglycemia in patients treated with insulin. Foundational to all such efforts is education of the patient (and also members of his or her family) regarding the symptoms of hypoglycemia and following the recognition of such symptoms, the urgency of the need to ingest a food or liquid rich in glucose, sucrose, or other rapidly digested form of carbohydrate; an example is provided by orange juice supplemented with sucrose (cain sugar). This baseline approach has been extended by the development of specific diabetes-oriented products, such as squeezable tubes containing an emulsion containing glucose in a form that can be rapidly absorbed through the mucous membranes of the mouth, throat, stomach, and small intestine. Preparations of the counter-regulatory hormone glucagon, provided as a powder, have likewise been developed in a form amenable to rapid dissolution and subcutaneous injection as an emergency treatment of severe hypoglycemia. Insulin pumps have been linked to a continuous glucose monitor such that subcutaneous injection of insulin is halted and an alarm is sounded when hypoglycemic readings of the interstitial glucose concentration are encountered. Such a device-based approach has led to the experimental testing of closed-loop systems in which the pump and monitor are combined with a computer-based algorithm as an “artificial pancreas.”
For more than three decades, there has been interest in the development of glucose-responsive materials for co-administration with an insulin analogue or modified insulin molecule such that the rate of release of the hormone from the subcutaneous depot depends on the interstitial glucose concentration. Such systems in general contain a glucose-responsive polymer, gel or other encapsulation material; and may also require a derivative of insulin containing a modification that enables binding of the hormone to the above material. An increase in the ambient concentration of glucose in the interstitial fluid at the site of subcutaneous injection may displace the bound insulin or insulin derivative either by competitive displacement of the hormone or by physical-chemical changes in the properties of the polymer, gel or other encapsulation material. The goal of such systems is to provide an intrinsic autoregulation feature to the encapsulated or gel-coated subcutaneous depot such that the risk of hypoglycemia is mitigated through delayed release of insulin when the ambient concentration of glucose is within or below the normal range. To date, no such glucose-responsive systems are in clinical use.
A recent technology exploits the structure of a modified insulin molecule, optionally in conjunction with a carrier molecule such that the complex between the modified insulin molecule and the carrier is soluble and may enter into the bloodstream. This concept differs from glucose-responsive depots in which the polymer, gel or other encapsulation material remains in the subcutaneous depot as the free hormone enters into the bloodstream. An embodiment of this approach is known in the art wherein the A chain is modified at or near its N-terminus (utilizing the α-amino group of residue A1 or via the ε-amino group of a Lysine substituted at positions A2, A3, A4 or A5) to contain an “affinity ligand” (defined as a saccharide moiety), the B chain is modified at its or near N-terminus (utilizing the α-amino group of residue B1 or via the ε-amino group of a Lysine substituted at positions B2, B3, B4 or B5) to contain a “monovalent glucose-binding agent.” In this description the large size of the exemplified or envisaged glucose-binding agents (monomeric lectin domains, DNA aptamers, or peptide aptomers) restricted their placement to the N-terminal segment of the B chain as defined above. In the absence of exogenous glucose or other exogenous saccharide, intramolecular interactions between the A1-linked affinity ligand and B1-linked glucose-binding agent was envisaged to “close” the structure of the hormone and thereby impair its activity. Only modest glucose-responsive properties of this class of molecular designs were reported (Zion et al., 2012).
The suboptimal properties of insulin analogs modified at or near residue A1 by an affinity ligand and simultaneously modified at or near residue B1 by a large glucose-binding agent (i.e., of size similar or greater than that of an insulin A or B chain), are likely to be intrinsic to this class of molecular designs. Indeed, the rationale for such designs relied on the low activity of insulin analogs containing short chemical cross-links between the α-amino groups of residues A1 and B1 but overlooked the native or enhanced activity of an insulin analogue containing a peptide linker between these residues of length similar to or exceeding that of an insulin A or B chain. Thus, the putative “closed” form of the above insulin analogues may not in fact be adequately constrained in conformation to provide significant impairment of receptor binding (relative to the modified insulin in the presence of exogenous glucose) and hence to provide useful or optimal glucose-dependent biological activity. The prescription of the above class of insulin analogues also overlooked the marked reduction in activity, irrespective of free glucose concentration, likely to arise on substitution of residues A2 or A3 (IleA2 or ValA3) by other aliphatic or non-aliphatic amino acids (such as Lysine); these analogues would be expected to have negligible biological activity and thus not be useful as is long known in the art. Binding of an insulin analogue to the insulin receptor would also be impaired, but to a lesser degree, by substitution of Lysine at position A1. Also overlooked in the above class of insulin analogues is potential advantages of an alternative type of glucose-regulated conformational switch that may not only affect affinity of the analogue for the insulin receptor, but also the extent of insulin self-assembly and its rate of disassembly as might pertain to glucose-regulated pharmacokinetic properties of the subcutaneous depot.
Surprisingly, we have found that a fundamentally different class of molecule designs may optimally provide a glucose-dependent conformational switch between closed and open states of the insulin molecule without the above disadvantages. The analogues of the present invention thus contain one or more saccharide modifications at or near the C-terminal end of the B chain rather than at or near the N-terminal end of the A chain (FIGS. 3A and 3B). Further, the analogues of the present invention avoid bulky glucose-binding agents at or near residue B1 and instead employ small chemical entities (phenylboronic acid derivatives; PBA) at or near residue A1. The closed state of the present invention, tethered by an interaction (either non-covalent or covalent but reversible) between a saccharide modification (or non-saccharide analogue with similar functional groups) at or near the C-terminal end of the B chain and a small chemical entity at or near the N-terminal end of the A chain, is thus different from and unrelated to that disclosed previously. The closed state of the present invention exploits a protective hinge in the insulin molecule that opens to engage the insulin receptor. Mini-proinsulins or single-chain insulin analogues in which a short covalent tether links the C-terminus of the B chain to the N-terminus of the A chain are known in the art to exhibit very low or undetectable activity (FIG. 2). The small size of the PBA moiety attached at or near the N-terminus of the A chain and its reversible binding to a cognate PBA-binding element at or near the C-terminus of the B chain would provide a conformational switch near to the classical dimerization surface of insulin and hence make possible glucose-regulation of insulin assembly and disassembly. The essence of this invention does not depend on the specific molecular embodiment of the modification of the B chain since PBA (and PBA derivatives as a fluoro-phenylboronic acid) exhibit reversible covalent bonding to diol functions of both (a) diverse saccharides (differing in the number and composition of the monosaccharide subunits) and also (b) non-saccharide organic compounds that present diol functions (or α-hydroxycarboxylate as alternative PBA-binding functions) that mimic those found within a monosaccharide (FIG. 3B).
The insulin analogues of the present invention, whose biological availability (modulated by glucose-regulated rates of hexamer disassembly in the subcutaneous depot) and/or biological potency (modulated by glucose-dependent affinity for the insulin receptor) would be stronger under conditions of hyperglycemia than under conditions of hypoglycemia, would enhance the general safety, efficacy, simplicity and convenience of insulin replacement therapy. Such an insulin analogue formulation would be compatible with multiple devices (such as insulin vials, insulin pens, and insulin pumps) and could be integrated with modifications to the insulin molecule known in the art to confer rapid-, intermediate-, or prolonged insulin action. In addition, the present glucose-regulated conformational switch in the insulin molecule, engineered between the C-terminus of the B chain and N-terminus of the A chain, could be combined with other glucose-responsive technologies (such as closed-loop systems or glucose-responsive polymers) to optimize their integrated properties. We thus envisage that the products of the present invention will benefit patients with either Type 1 or Type 2 diabetes mellitus both in Western societies and in the developing world.